The elucidation of the roles of chemokine receptors in entry of HIV and pathogenesis has allowed insights into the disease and possible points of therapeutic intervention. The identification of naturally occurring polymorphisms of the CCR5 gene has shown that gene disruption provides protection from CCR5 using viruses. Since these polymorphisms are present in otherwise healthy appearing individuals, genetic intervention strategies that prevent or limit expression of CCR5 might provide protection from initial infection and possibly limit viral spread. We have demonstrated that intracellular expression of a CCR5- specific single-chain antibody (i.e., intrabody), containing endoplasmic reticulum retention signals efficiently disrupted surface expression of CCR5. Intrabody-expressing cell lines were highly refractory to challenge with free HIV-1 or HIV-infected cells. We propose that HIV vector delivery of CCR5 and CXCR4 intrabody genes to selected hematopoietic cell populations will limit HIV entry. Our studies will investigate this approach to limit HIV infection of myeloid cells and T cells. In Specific Aim 1 we will investigate the in vitro virological and biological consequences of disrupting cell surface expression of CCR5 and CXCR4 in CD4 T cells, macrophages and dendritic cells through HIV vector delivery of CCR5 and CXCR4 intrabody genes, respectively. In Specific Aim 2 we evaluate whether HIV vector delivery of CCR5 intrabody genes to CD34+ stem cells promotes and maintains disruption of CCR5 throughout myeloid differentiation in human-NOD/SCID mice. In Specific Aim 3 we will use SCID-hu mice to investigate whether thymocytes arising from CD34+ cells transduced with HIV vectors delivering CCR5 intrabody genes maintain intrabody expression. In Specific Aim 4 we will use human PBL-NOD/SCID mice to determine the biological and virological consequences of disrupting CCR5 in mature T cells by using an HIV vector to deliver CCR5 intrabody genes. Lastly, our proposed studies will contribute basic and pre-clinical insights into the feasibility of using HIV vectors to deliver and maintain ectopic gene expression in hematopoietic cells.